Inducible methods for repressing gene function

a technology of gene function and inducible methods, which is applied in the field of inducible methods for suppressing gene function, can solve the problems of loss of function of encoded gene product, many limitations in the genetic approach, and exceedingly difficult study of essential genes, required for cell viability, and purely genetic approach

Inactive Publication Date: 2003-06-10
PRESIDENT & FELLOWS OF HARVARD COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The mutation involved is frequently a change in the gene's sequence which results in a loss-of-function of the encoded gene product.
Unfortunately the genetic approach has many limitations.
Indeed the study of essential genes, required for cell viability, is exceedingly difficult using a purely genetic approach.
Nevertheless, the exact biological processes in which the essential gene is involved are difficult to determine due to the inability to isolate and/or study the doomed knockout yeast spore or the inviable homozygous mouse zygote.
Thus the downstream effects on specific aspects of cell function following removal of the essential gene product cannot be readily determined using these traditional "knockout" st...

Method used

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  • Inducible methods for repressing gene function
  • Inducible methods for repressing gene function
  • Inducible methods for repressing gene function

Examples

Experimental program
Comparison scheme
Effect test

example 1

5.1 Example 1

Use of Double Shutoff Yeast Strain Containing Copper-Inducible Yeast Alleles of Both ROX1 And UBR1 to Determine that TBP-Associated Factors are not Generally Required for Transcriptional Activation in Yeast

Materials and Methods

The parent strain ZMY60, containing copper-inducible alleles of UBR1 and ROX1, was created as follows. A cassette containing the copper-inducible derivative of the HIS3 promoter (Klein, C and K. Struhl (1994) Science 266: pp. 280-282) and 2 kb of upstream flanking sequence was inserted at the initial ATG of a plasmid-borne genomic fragment of ROX1 to create the URA3 integrating plasmid ZM195. The same cassette was inserted at the initial ATG of UBR1 to create ZM197. Both of these copper-driven alleles were introduced into yeast strain KY114 (Iyer, V and K. Struhl (1995) Mol. Cell. Biol. 116: pp. 7069-7086) in successive two-step gene replacements. To create TAF disruption molecules, another cassette comprising an inframe fusion of ubiquitin, argin...

example 2

5.2 Example 2

The following example demonstrates how any given target gene can be configured for the double shutoff system in Saccharomyces cervisiae. The system requires two basic components: first, a parent strain containing copper-inducible alleles of both ROX1 and UBR1; and second, a short 5' fragment of the target gene of interest fused in frame to a ubiquitin-arginine-lacI-HA ("URLF") cassette and driven by the ANB1 promoter.

The strain ZMY60 has the genotype: MAT ay, ACE-UBR1, Ace-ROX1, trpl-D1, ura3-52Z LEU2, HIS3, ade2-101 in a KY114 background. To generate a parent strain in a different background, one utilizes the URA3 integrating plasmids ZM195 and AM197, which must be used in successive two-step gene replacements to generate a parent strain containing copper-inducible ROX1 and UBR1. To create double-shutoff parent strain, one integrates ZM195 into a desired strain with AflII. The URA3 marker is the loopout on FOA (5-fluorotica acid). One would then check for correct loopo...

example 3

5.3 Example 3

DKO strains and constructs of TAF19 (strain ZMY67), TAF60 (ZMY66), TAF90 (ZMY68), TAF 130 (ZMY69, TBP, AND TFIIB (ZMY71).

Strain ZMY60 (the DKO parent strain)

Useful Saccharomyces cervisiae Strains

Parent Strains

ZMY59 (Ace-UBR1 only strain)

ZMY61 (Ace-ROX1 only strain)

ZMY103 (ZMY60 his3.DELTA.200)

ZMY117 (ZMY60 leu2::PET56)

ZMY118 (ZMY60 his3.DELTA.200, leu2::PET56)

Shutoff Strains

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Abstract

Methods for the rapid repression of gene function in eucaryotic cells are disclosed including inducible means for both shutting down a targeted gene's transcription and rapidly removing a targeted gene's polypeptide product.

Description

1. BACKGROUND OF THE INVENTIONGenetics is essentially an approach to understanding biological processes through the systematic elimination of gene function. Historically, the genetic approach has involved the development of "screens" for mutations in genes which affect a specific phenotypic trait of an organism. The great advantage of this approach has been that no prior knowledge of the molecular nature of the genes involved is required because the "screen" identifies the affected genes by marking them with mutations. The mutation involved is frequently a change in the gene's sequence which results in a loss-of-function of the encoded gene product. Unfortunately the genetic approach has many limitations. Indeed the study of essential genes, required for cell viability, is exceedingly difficult using a purely genetic approach. As the famous molecular biologist David Botstein once put it, "death is not a phenotype;" an expression which encapsulates the frustration of attempting to st...

Claims

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Application Information

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IPC IPC(8): C07K14/00C12N15/67C12N15/63C12N15/85C12N15/12
CPCC07K14/00C12N15/635C12N15/85C12N15/67C07K2319/00C12N2830/702C12N2830/002C12N2830/005
Inventor STRUHL, KEVINMOQTADERI, ZARMIK
Owner PRESIDENT & FELLOWS OF HARVARD COLLEGE
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